Selenium (Se) is a highly promising compound for the chemoprevention of prostate cancer. Although intensively studied, the molecular mechanisms responsible for the chemopreventive effectiveness of Se remain unclear. We hypothesize that a loss of redox-protective activity in the prostate results from the functional suppression of key redoxselenoproteins (RoxSePs; e.g. glutathione peroxidase, GPx and thioredoxin reductase, TxR). This process occurs during aging or low dietary Se intake leaving prostate tissues defenseless against the oxidative damage responsible for carcinogenesis. To test this hypothesis, we will produce recombinant mouse models that lack factors involved in co-translationally generating selenoproteins. Selenoproteins are co-translationally formed when other proteins that recognize selenocysteine insertion sequences (SECIS) insert selenocysteine at opal stop codon (UGA) positions instead of terminating the protein chain. One such protein, SECIS binding protein 2 (SBP2), is our primary target. The SBP2 gene will be targeted to simultaneously eliminate all selenoproteins, this will avoid compensation by multiple RoxSeP isoforms and functional overlap by various RoxSePs. Standard targeting methods are unlikely to work since selenocysteyl-tRNA gene knockout (KO) mice (-/-) exhibit embryonic lethality. Lethality, however, can be overcome by controlling tissue-specific targeting and KO timing. To achieve these goals we will use a specialized Cre/Iox recombination system involving Cre-ERT. In the Cre-ERT fusion protein, Cre is fused with a mutated estrogen receptor ERT that is active only when 4-hydroxytamoxifen (4-OH-T) is bound and then excises DNA between adjacent Iox sites. The prostate-tissue-specific probasin (PB) promoter will be inserted in front of the Cre-ERT molecule to target SBP2 only in the mouse prostate. We will use the resulting 4-OH-T inducible PB-Cre-ERT/floxed SBP2 (Iox-SBP2-lox) mice to study selenoprotein function during early and late-stage prostate carcinogenesis. Aim 1: Will isolate and characterize the SBP2 gene. An SBP2 cDNA clone will be used to screen a mouse 129S genomic library. Sequencing will be performed to determine the intron-exon boundaries and the most useful exon for targeting. Aim 2: Will generate recombinant mice to study the role of SBP2 in the prostate. The PB-Cre-ERT/floxed SBP2 mice generated will be given 4-OH-T at the appropriate stage to induce recombinant excision of SBP2. Aim 3: Will examine the SBP2 (-/-) mouse phenotype and the role of SBP2 gene loss during carcinogenesis. Prostate cells containing PB-Cre-ERT/floxed SBP2 will be placed into tissue-culture and examined for the lack of 75Se isotope incorporation into GPx, and TxR using antibodies generated in our lab. These animals are needed to perform pre-clinical evaluation of promising chemopreventive selenocompounds in the presence or absence of RoxSePs during prostate carcinogenesis.